Alone among the malaria species which infect humanity, Plasmodium falciparum causes the erythrocytes which it invades to sequester in the deep vascular beds of various tissues. This sequestration phenomenon is observed in peripheral blood smears by the presence of immature (non-adherent) ring-stage parasitized erythrocytes and the absence of mature (adherent) trophozoite and schizont stage parasites, the latter having localized to postcapillary venules (Bignami and Bastianeli (1889) Reforma Medica 6: 1334-1335) (All documents cited herein supra and infra are hereby incorporated by reference). Sequestration allows the parasite to develop in a microenvironment of low oxygen tension and to evade splenic immune surveillance (Langreth and Peterson (1985) Infect. Immun. 47: 760-766).
Faliparcum malaria can have protean manifestations, ranging from asymptomatic infection, to mild disease (symptoms may include fever, arthralgias, abdominal pain, diarrhea, headache, nausea, fatigue and others in various combinations), to severe disease (recognized severe forms include cerbral malaria with coma, pulmonary edema with consequent resperiatory failure, severe anemia with consequent hemodynamic/cardiopulmonary decompensation) often resulting in death. These symptoms of severe malaria resulting in vast mortality worldwide are believed to be imparted by sequestration (Warrell et al. (1990) Trans. Soc. Trop. Med. Hyg. 84:1-65). Owing to the devastating consequences of the disease, and the potential for therapeutic intervention, researchers have long sought to isolate the parasite protein(s) responsible for the cytoadherence of P. falciparum infected erythrocytes (IRBC) to postcapillary venular endothelium.
Cytoadherence appears to be a complex event, with multiple binding phenotypes displayed by both culture-adapted and wild-type IRBC isolates. In vitro models demonstrate that different cell lines, such as C32 amelanotic melanoma cells (Schmidt et al. (1982) J. Clin. Invest. 70: 379-386), human umbilical vein (Udeinya et al. (1981) Nature 303: 429-431) and human microvascular endothelial cells (Johnson et al. (1993) J. Infect. Dis. 167: 698-703) support adhesion of IRBC. More recently, with the availability of purified molecules, an array of endothelial ligands such as CD36 (Ockenhouse et al. (1989)Science 243: 1469-1471) ICAM-1 (Berendt et al. (1989) Nature 341: 57-59) VCAM-1 (Ockenhouse et al. (1992) J. Exp. Med. 176: 1183-1189), E-selectin (Ockenhouse et al., 1992), and the extracellular matrix molecules thrombospondin (Roberts et al., year, Nature 318: 64-66) and chondroitan sulfate A (Rogerson et al. (1995) J. Exp. Med. 182:15-70) have demonstrated the capacity to bind IRBC. While culture-adapted IRBC can be selected to bind each of these ligands, only CD36 is able to bind nearly all wild-type parasite strains isolated in the field (Ockenhouse et al (1991) Proc. Natl. Acad. Sci. U.S.A. 88:3175-b 3179); Hansen et al (1990) Blood 76: 1845-1852). Furthermore, P. falciparum parasites which no longer cytoadhere in vitro to cells expressing CD36, are unable to establish a virulent infection in primates, suggesting the primary role of sequestration for parasite survival (Langreth and Peterson, 1985, supra).
Electron microscopy studies have demonstrated that IRBC adherence to endothelium occurs along electron-dense protrusions, called xe2x80x9cknobsxe2x80x9d, on the erythrocyte surface (MacPherson et al. (1985) Am. J. Pathol. 119: 385-401). The IRBC surface ligands for both CD36 and thrombospondin receptors have been localized to these knobs (Nakamura et al. (1992) J. Histochem. Cytochem. 40:1419-1422). Although culture-adapted laboratory parasites may bind CD36 in the absence of surface knobs, (Udomsangpetch et al. (1989) Nature 338: 763-765) the prevailing view is that the CD36-binding protein(s) localizes at the surface of the knob of all wild-type parasites.
CD36, an 88 kD glycoprotein expressed on the surface of microvascular endothelium, platelets, and monocytes belongs to a family of related proteins containing extensive amino acid homology (Greenwalt et al. (1992) Blood 80:1105-1115; Calvo et al. (1995) Genoinics 25: 100-106). Also known as platelet glycoprotein IV or IIIb, CD36 is expressed in a regulated fashion during cell development (Abumrad et al. (1993) J. Biol. Chem. 268: 17665-17668) and its expression is modulated by cytokines (Huh et al. (1995) J. Biol. Chem. 270: 6267-6271; Johnson et al. (1993) J. Infect. Dis. 167: 698-703). CD36 has binding sites for several molecules involved in hemostasis and atherogenesis, including collagen (Tandon et al. (1989) J. Biol. Chem. 264: 7570-7575), thrombospondin (Asch et al. (1992) Biochem. Biophys. Res. Common. 182: 1208-1217), oxidized low density lipoprotein (LDL) (Endemann et al. (1993)J. Biol. Chem. 268: 11811-11816), long chain fatty acids (Abumrad et al. (1993) supra), and anionic phospholipids (Rigotti et al. (1995)J. Biol. Chem. 270: 16221-16224).
Cytoadherence of IRBC in vitro can be inhibited or reversed by antibodies directed against either the surface of parasitized erythrocytes or against endothelial cell ligands. IRBC binding to CD36 is blocked by monoclonal antibodies OKM5 (Barnwell et al. (1985) J. Immunol. 135:3494-3497) and OKM8 (Ockenhouse et al., 1991) directed against discontinuous epitopes but not by other CD36 monoclonal antibodies which recognize linear continuous epitopes (Ockenhouse et al., unpublished observations) establishing the importance of conformationally-correct protein structure for IRBC binding to CD36. In monkeys, sequestration can be reversed by passive transfer of hyperimmune sera, leading to the clearance of IRBC in the spleen (David et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80: 5075-5079).
A number of parasite-derived and altered host proteins have been postulated to mediate IRBC cytoadherence. PfEMP1, a large molecular weight, size variable protein which is the product of the var gene family involved in malaria antigenic variation (Baruch et al., 1995; Su et al. (1995) Cell 82: 89-100; Smith et al., 1995), has characteristics which suggest its involvement in cytoadherence: PfEMP1 is expressed on the external erythrocyte surface as demonstrated by radioiodination and immunofluorescence; PfEMP1 is readily cleaved from the IRBC surface by proteolytic enzymes such as trypsin at concentrations known to abolish IRBC adhesion, and PfEMP1 varies its size in a manner which correlates with changes in both strain specificity and IRBC adhesion (Leech et al. (1984) J. Exp. Med. 159: 1567-1575; Baruch et al., 1995). Another candidate, band 3, is a surface protein of normal human erythrocytes which has been shown to be altered by malaria infection, exposing cryptic peptides in loops 3 and 7 (Crandall and Sherman (1994) Parasitol. 108:389-396). Monoclonal antibodies against altered band 3 inhibit cytoadherence in vitro (Crandall and Sherman, 1994) and synthetic peptides based on the cryptic epitopes of band 3 affect sequestration in vivo (Crandall et al. (1993) Proc. Natl. Acad Sci. U.S.A. 90:4703-4707).
A third candidate cytoadherent ligand, Sequestrin, was identified by immunoprecipitation of a high molecular weight ( greater than 200 kD) IRBC surface protein by anti-idiotype antibodies raised against the CD36-specific monoclonal antibody OKM8 (Ockenhouse et al., 1991). These monospecific, anti-idiotypic antibodies bound to the surface of IRBC and inhibited IRBC adhesion to CD36.
Cytoadherence, then, is critical to both the survival (Langreth and Peterson (1985) Infect. Immun. 47: 760-766) and pathology (Warrell et al., supra) of P. falciparum parasites, and is a multi-faceted process, involving a number of endothelial receptors, and one or more IRBC counter-receptors. Identifying IRBC counter-receptors has been an elusive goal of malaria research, possibly due to antigenic variation, immunologically cross-reactive proteins, or other mechanisms by which the parasite masks the presence of biologically vital adhesion protein(s) on the cell membrane. As well, the modalities for demonstrating an adhesion protein can be misleading; for example, an antibody which identifies a non-cytoadherent malaria protein can still inhibit adherence in a binding assay by causing IRBC agglutination.
The identification of an IRBC counter-receptor would allow for the design of anti-sequestration and anti-malaria agents, drugs and vaccines necessary for the prevention of cerebral malaria and the reduction of mortality from this disease worldwide.
The complexity of the cytoadherence process led us to conclude that proof of a parasite-expressed counter-receptor must include binding of purified forms of the putative counter-receptor to a receptor, as well as competitive inhibition of IRBC binding to an endothelial receptor by the putative counter-receptor. In the absence of this direct evidence, data implicating a protein in cytoadherence can only be considered suggestive. We further employed this idea in our approach to isolating the Sequestrin gene, by using radiolabelled CD36 as a probe to identify clones from a P. falciparum cDNA expression library.
While it is theoretically possible that the use of anti-idiotypic antibody could be used to immunopurify Sequestrin from a lysiate of P. falciparum parasites, this approach is not technically feasible. Numerous approaches have been tried over the years to isolate the cytoadherence receptor. The approaches used have utilized antibodies to screen a cDNA library of P. falciparium. However, since a number of laboratories with many personnel have succeeded in isolating cDNA clones using this approach, they have been uniformly unsuccessful in isolating the parasite cytoadherence receptor. This is primarily because the parasite is promiscuous in the sense that many of the parasite""s proteins cross react with each other because of similar amino acid motifs and redundant amino acid repeat units. Therefore, it is unlikely that the use of antibodies, including the use of anti-idiotype antibodies, to probe a cDNA library will yield a clone with properties for which one is searching. Two recent groups of researchers searching for the cytoadherence gene/protein have come up with alternative proteins (Pasloske et al. (1993) Mole. Biochem Parasitol. 59: 59-72; Barnes et al. (1995) Exp. Parasitol 81: 79-89). Due to these failures, we reasoned that the use of antibodies, whether anti-idiotypic or not, would not yield a positive malaria clone responsible for the binding to CD36. Instead, we used expression-cloning which utilizes receptor-counter-receptor recognition. In this approach we used CD36 to probe a cDNA library to isolate only those clone(s) which bind to CD36 thereby circumventing the possibility of getting a cross-reactive protein via the antibody selection technique. Only a single clone from 70,000 clones was isolated thus confirming the infrequency of positive selection utilizing expression-cloning yet confirming functional importance when the clone was sequenced and the Sequestrin protein expressed.
We report here direct evidence that Sequestrin is a high affinity receptor for CD36, and is distinct from both PfEMP1 and eythrocyte band 3. By expression cloning, we have obtained 2 kB of the nucleotide sequence for Sequestrin, and have used recombinant fusion protein studies to determine the CD36-binding domain of the protein.
Sequestrin is present and expressed in P. falciparum isolates which bind to CD36. Sequestrin is present in the genome of P. falciparum isolates which do not bind CD36; it is expressed in lower amounts, if at all, in laboratory isolates which do not bind, and levels of expression in field isolates which do not bind CD36 is currently unknown. Sequestrin is not present in the genome of P. vivax (as determined by PCR detection using primers based on the Sequestrin sequence). Therefore, DNA-based methods which detect Sequestrin in the genome of the parasite would be a technique to diagnose infections with P. falciparum malaria.
Therefore, it is an object of the present invention to provide a Sequestrin cDNA fragment encoding 1958 nucleotides useful as a diagnostic agent, a therapeutic agent, and a DNA-based vaccine.
It is another object of the invention to provide an amino acid sequence for Sequestrin protein encoding 652 amino acids.
It is another object of the invention to provide a recombinant vector comprising a vector and the above described DNA fragment.
It is a further object of the present invention to provide a host cell transformed with the above-described recombinant DNA construct.
It is another object of the present invention to provide a method for producing Sequestrin which comprises culturing a host cell under conditions such that the above-described DNA fragment is expressed and Sequestrin protein is thereby produced, and isolating Sequestrin protein for use as a vaccine and a diagnostic agent, and a therapeutic agent.
It is still another object of the invention to provide a purified Sequestrin protein useful as a vaccine against malaria and for detecting the presence of said disease in a suspected patient.
It is a further object of the present invention to provide an antibody to the above-described recombinant Sequestrin protein.
It is yet another object of the invention to provide a malaria vaccine effective for the production of antigenic and immunogenic response resulting in the protection of a mammal against malaria.
It is yet another object of the present invention to provide a method for the diagnosis of malaria comprising the steps of:
(i) contacting a sample from an individual suspected of having the disease with antibodies which recognize Sequestrin; and
(ii) detecting the presence or absence of a complex formed between Sequestrin and antibodies specific therefor.
It is a further object of the present invention to provide a diagnostic kit comprising a recombinantly produced Sequestrin antibody and ancillary reagents suitable for use in detecting the presence Sequestrin in mammalian tissue or serum.
It is yet another object of the present invention to provide a method for the diagnosis of malaria from a sample using the polymerase chain reaction, said method comprising:
(I) extracting RNA from the sample;
(ii) converting the RNA into complementary DNA;
(iii) contacting said DNA with
(a) at least four nucleotide triphosphates,
(b) a primer that hybridizes to Sequestrin DNA, and
(c) an enzyme with polynucleotide synthetic activity,
under conditions suitable for the hybridization and extension of said first primer by said enzyme, whereby a first DNA product is synthesized with said DNA as a template therefor, such that a duplex molecule is formed;
(iv) denaturing said duplex to release said first DNA product from said DNA;
(v) contacting said first DNA product with a reaction mixture comprising:
(a) at least four nucleotide triphosphates,
(b) a second primer that hybridizes to said first DNA, and
(c) an enzyme with polynucleotide synthetic activity,
under conditions suitable for the hybridization and extension of said second primer by said enzyme, whereby a second DNA product is synthesized with said first DNA as a template therefor, such that a duplex molecule is formed;
(vi) denaturing said second DNA product from said first DNA product;
(vii) repeating steps iii-vi for a sufficient number of times to achieve linear production of said first and second DNA products;
(viii) fractionating said first and second DNA products generated from said Sequestrin DNA; and
(ix) detecting said fractionated products for the presence or absence of Sequestrin in a sample.
It is yet another object of the present invention to provide a method for the detection of Sequestrin in a sample which comprises assaying for the presence or absence of Sequestrin RNA or DNA in a sample by hybridization assays.
It is an object of the present invention to provide a therapeutic method for the treatment or amelioration of symptoms of malaria, said method comprising providing to an individual in need of such treatment an effective amount of a Sequestrin antibody or an agent which inhibits Sequestrin expression or function in a pharmaceutically acceptable excipient.